Alkyl pyridinium salt, 2-carboxaldehyde oximes and process of preparation



.311! orga ic sulfate, and -so on. tablrshed whether this new-class hasthe synorthe anti- Configuration.

United States Patent Anna-m- PYRIDINIUMW SALT; z-oARBoxALDE- oxnnss- ANDPRGGESS: on PREPARA- 1 Tron and David Nticlinians'ohn, New York} N. ii,assignors tdfthdUiiitd 'St'ates or Amene'aas represented-pyrite sereniy-mumm Ne firmin Application Juli 10,1956,

Serial No. 597,055

10. Claims. or. 260-296 The present invention provides new compositionsof matter which have been found to have antidotal properties for theirreversible inhibitors of cholinesterase.

The'cll e nical lesion in alkylphosphate intoxication is I theinhibition of acetylcholinesterase, although in some receptor; Quee-this causal connectionhad been. estab- 'lisfli'edit' be ame importantto" understandthe' exact reaction mechanism between acetylcholinesteraseand alkylphosphates. Studies based on the molecular forcesacting-betweenaeetylcholine and theenzyrneledto anunder- This analysismade it standing of the hydrolytic process. poss'ibleto elucidatetheexact mechanism of-the reaction between alkylphosphates and enzyme.

According to these studies,-in' the physiological action ofacetyldholinesterase an acetyl enzyme is formed which then reacts with H010 form acetate and enzyme. In

the reactions with alkylphosphate a phosphorylated enzyine is ier rnedwhieh, however; does net-reactqwitnlrl o; the ph'esphoty-l g roupzremains attachedi toz'the enzyme and-the: ens nie is inhibiteec since itis a vital enzyme fer aer efunetiemdeatn ensues.

i a e antluote has been developed on the rerniseythat "'nneleopnilieagents should be able to dephos'pliorylate the enzyme and thereby repairthe chemical lesion. Hy-

ACet Ie'heIinesteraSe shows a specificity toward fete, that the:activitw ofa hyarexylamin derivative effective" as a specific antidoteagainst lethal alkylphosj phate intoxication:-

where R represents H or alkyl and R" repi'esentsjmethyl tonethyl groups.These compounds are saltswheret'he anionic part-(X5) is not critical andcan be a halogenide,

It has notyet been es- Preparation of these compounds can be achievedusing; two. general methods:

I my action of alkyl halides, sulfates, toluene sul- .fonats or otherN-methylating. agents on. tertiary pyridineoximes, or,

(b) By action of hydroxylamineons quaternary. aldehydes and ketones.

'atroorn temperature overnight. ml. acetone and filtered and rinsed withacetone. The

Tertiary pyridine oximes can be prepared in the general way, either byreacting aldehydes or ketones with hydroxylamine at slight acid oralkaline pH or by action of nitrous acid or its esters on pyridinecompounds containing active methylene groups, or by reaction between apyridine nucleus andmercuric fulminate or by interaction of diazoniumsalts with formaldoxime or acetaldoxime. The subsequentalkylation can bedone either at roomor elevated temperatures with or without solvents.

The quaternary aldehydes are prepared byapplying methylating agents toaldehydes or ketones or by,prepaiingquaterna'ryderivatives containingactive methylene groups and reacting these with nitrous acidderivatives, e. g. p-nitroso-dirnethyl aniline and subsequentsaponification. 7

Reactiouofthese aldehydes or ketones with hydroxylamineistefiectediinaqueous or alcoholic solutions at temperatures between 0and C. and neutral or slightly acidic" pH'.

" Briort'o thepresent invention, the'class of oximes'containing'japositivelygcharged nitrogen atom of the general formula, aspreviously-shown, was not known so that the novel process of "thepresent invention includes the synthesis'of these compounds.

The procedures "are further illustrated by. the following exampleswithout being restricted thereto:

EXAMPLEItZ-PYRIDINE ALDOXIME METI-IIO- DIDE) Z pyridi'ne'aldoxime 3.05g., nitrobenzene 10 ml., and

This is mixed with 40 yieldis'" 88* percent of theory: Recrystallizationfrom methanol produced bright yellow needles having a melting.pointof224-22S C It is very soluble in water, fairly soluble in hotalcohols, poorly soluble in cold alcoholsand insoluble in ether andacetone.

EXAMPLE- II (.4-PYRIDINE ALDOXIME METHIOF DIDE); w I t-pyridinealdoxime, 1.22"g., was' dissolvedin acetone 20 ml and methylioc'lide,ml., was added! The;quaternary'salt started to precipitatevery shortly.After several hoursif was filtered on and washed with acetone. The yieldwas 99 percent of theory, forming bright yellow crystals having amelting point of 181-183 C.

EXAMPLE III (METHYL-2-PYRIDYL KETOXIME METHIODIDE) The quaternary ketonewas prepared from methyl-2- pyridyl ketone with excess of methyliodidein acetone at room temperature producing faint yellow crystals having amelting point of 163--l64 C. This ketone, 6.6 g., was reacted withaqueous solution of NH OHHCI, 2.6 g., neutralized with NaOH to pH 6-7.After testing for 15 minutes onstearn: bath the waterwas evaporatedunder reduced:- pressure and. the almost dry residue extracted with hotabsolute "ethanol: to eliminate mineral salts; On coolingntheoximecrystallized. The overall yield was .45 percent, producing yellowneedles having a melting 'point 05213-214 7 Certain phosphate esterssuch as tetraalkylpyrophosphates diallryl-prnitrophenyl phosphates,andrdialkyl fluorophosphates; are potent irreversible inhibitors ofacetylcholinesterase and esterases in general. The reactivation ofalkylphosphate-inhibited acetylcholinesteraseis of both practical andtheoreticalimportance. It is of practical interestbecause-themost potentchemical warfare gases and some'powerful insecticides arealkylphosphates and their lethal action is due to the inhibition ofacetylcholi'nesterase. It is of theoretical interest because the'meclnanism of inhibition and of reactivation is very closely related to themechanism of enzymic hydrolysis. This enzyme contains two sites, (i) ananionic site which contributes to the catalytic activity by binding andorienting molecules containing substituted ammonium structures, and (ii)an esteratic site which interacts with the ester function and isprimarily responsible for the hydrolytic activity. During the hydrolysisof a carboxylic ester a basic group in the esteratic site is acylated toform an acyl-enzyme as intermediate. Acetyl-enzyme (from acetate-estersor anhydrides) rapidly reacts with water to produce acetic acid and toregenerate the free and active enzyme. The alkyl phosphate inhibitorsreact with the same basic group to form a dialkyl phosphoryl enzymewhich however reacts only very slowly with water.

The inhibitory reaction is illustrated for a fiuorophosphate:

G II I H-Q (R)2PF (R0)=P=O HF Active enzyme Inhibitor Inhibited enzymewhere H-G represents the esteratic site containing an acidic group (H)and a basic group Theory predicts that nucleophilic reagents shoulddephosphorylate the enzyme and thus restore its activity. When R=ethyl(inhibitor=diethyl fiuorophosphate or tetraethyl pyrophosphate (TEPP))reactivation is readily accomplished by a large number of suitablecompounds. When R=isopropyl (inhibitor: diisopropyl fluorophosphate(DFP)) reactivation is more diflicult.

Experiments show that the anionic site survives the inhibition of theenzyme and can contribute to the reactivation process. Therefore a verygood reactivator might results it appeared that measurements with TEPPmight not be rate measurements but rather the extent of reactivationachieved at equilibrium of the reaction:

Postulated phosphorylated oxime Inhibited Reactivator enzyme Activeenzyme To test this possibility the inhibited enzyme was diluted 400times (instead of 7.5 times) before the reactivator was added. Underthese circumstances the postulated phosphorylated oxime should begreatly reduced and the equilibrium displaced to the right. The resultsso obtained, Table 113, are consistent with the assumption ofequilibrium. High reactivations are obtained with very low reactivatorconcentrations. With TEPP even at these low concentrations it appearsthat the rate is much less than 1 minute and that the measurementsconstitute equilibrium values. The DFP data seem to indicate a rate, butthe situation is not clear.

The quaternary oxime is a million times better than the non-methylatedcompounds and 50,000 times better than picolinohydroxamic acid inreactivating TEPP inhibited enzyme. The reactivation appears to beapproaching enzyme speeds.

Table I.-P ercent reactivation of alkylphosphate-inhibitedacetylcholinesterase with Z-pyridine aldoxime methiodide Thereactivations were carried out as follows: 0.2 ml. of enzyme solutionprepared from Electrophorus electricus was treated with 0.01 ml. of TEPPor DFP solution (20 'y/ml.), and diluted after 1 hour in the cold to 1.5This solution was used as stock for reactivation; to 0.2 ml. were added0.2 ml. of reactivator solution of suitable concentration in 0.015 Mphosphate buffer (pH 7) and 0.007 M EDTA. After suitable incubations(1', 5, 11) the reactivated solution was diluted to 50 ml. with waterand 1 ml. was added to the manometric vessels for assay. The totalenzyme dilution was 5625, fold. In part B the inhibited enzyme wasdiluted to 80.0 ml. instead of 1.5 ml.

A. DILUTED 7.5 TIMES Most reactivators react directly with TEPP and DFP.This is also the case with the quaternary oxime. The rate as judged byacid production was fairly rapid but not extroardinary; with 0.01 Moxime and 0.002 M phosphate anhydride the time for 50% reaction at 25 C.and pH 7.4 was 12 and 20 minutes for TEPP and DFP respectively.

The nicotinoand picolinohydroxamic acids proved already to be of valueas antidote of the so-called nerve gas in animals. The quaternary oximeis thousands of times more potent as a reactivator. It appeareddesirable,

therefore, to investigate whether this compound was sufi'icientlynontoxic to be used as an antidote and whether,

if so, it would in fact serve as an antidote for the irreversibleinhibitors of cholinesterase. White mice weighing about 20 g. were usedwithout regard to sex. The general procedure was to inject paraoxonsubcutaneously followed 12 min. later by interperitoneal injection of 2-pyridine aldoxine methiodide (Z-PAM). In some cases multiple injectionsof 2-PAM were used starting l-2 min. before the paraoxon, repeated 5 or10 min. after the poison and in some cases again 15 min. later. Thedoses of paraoxon designated as LD and LD correspond to acute poisoning;death occurs in 5-20 min.

The acute toxicity of 2-PAM is given in Table H. The toxicity of thecompound, while important, is not especially great. Death which is dueto respiratory failure occurs within 10-20 min.

On the basis of the minimal lethal dose it was decided to use as amaximum dose 75 mg. 2-PAM/kg. This was considered to be a safe dose. Theantidotal properties of 2-PAM as seen from Table III are quite marked.Complete survival was obtained from a LD dose of paraoxon with a safedose of 2-PAM. Because of uncertainty concerning the time course of aneffective antidote level, it was thought that better results might beobtained with very low antidote doses if they were given as multipleinjections. As will be seen, three injections totaling only 11 nag/kg.save 7 out of 11 mice.

Table 1II.-Antidotal properties of Z-PAM In the case of multipleinjections of antidote the times are indicated in parenthesis. In othercases the antidote was added (I. P.) 1-2 min. after the poison (S. C.)

Paraoxon dose (mg/kg.) 2-PAM dose (mg./kg.) dead alive One mouse ofthese five died before the second injection could be i ivo mice of thesefour died before the third injection could be given Many inhibitors ofcholinesterase produce the same phosphoryl enzyme as paraoxon. However,this is not enough to insure that a quaternary oxime will be aneffective antidote for all these compounds because it is also necessarythat the quaternary oxime be able to penetrate to the same vital nervouscenters as the poison. One cannot predict, therefore, with certaintythat the quaternary oxime will be an effective antidote for all poisonsof this class, but it does appear likely that it will serve for a goodnumber of them. Since 2-PAM is a much poorer reactivator of the serumesterase, in vitro, it may be concluded that probably onlyacctylcholinesterase has been reactivated in these in vivo experiments.

It will be understood from the foregoing description that, while itpresents in detail the preferred procedural steps in sequential detail,it will be apparent that the invention is intended to embrace within itsscope such equivalent procedures as may become suggested to one skilledin the art as being operational under ambient or procedural conditionsat variance with the specifically described conditions in various ways;and accordingly, it will be understood that it is intended and desiredto embrace within the scope of this invention such modifications-andchanges as may be necessary or desirable to adapt it to varyingconditions and uses, as defined in the appended claims.

Having thus described our invention, what we claim as new and wish tosecure by Letters Patent is:

1. As a new composition of matter, a new class of oximes of the generalformula wherein R is a member selected from the group consisting of Hand lower alkyl and R" is a radical selected 6 from the group consistingof methyl and ethyl groups and X represents the anionic part of the R"Xsalt.

2. As a new composition of matter, 2-pyridine aldoxime methiodide.

3. As a new composition of matter, 4-pyridine aldoxime methiodide.

4. As a new composition of matter, methyl-Z-pyridyl ketoxime methiodide.

5. A process for producing 2-pyridine aldoxime methiodide, comprisingalkylating 2-pyridine aldoxime with methyliodide.

6. A process for producing 4-pyridine aldoxime methiodide, comprisingalkylating 4-pyridine aldoxime with methyliodide.

7. A process for producing a new class of oxirnes of the general formulawherein R is a member selected from the group consisting of H and loweralkyl and R" is a radical selected from the group consisting of methyland ethyl groups and X represents the anionic part of the RX salt, whichcomprises alkylation of tertiary pyridine oXimes with N-methylatingagents.

8. A process for producing a new class of oximes of the general formulaOR=\"OH A R X wherein R is a member selected from the group consistingof H and lower alkyl and R" is a radical selected from the groupconsisting of methyl and ethyl groups and X represents the anionic partof the R"X salt, which comprises reacting a quaternary pyridine ketonewith hydroxylamine.

9. The process claimed in claim 8 wherein the quaternary pyridine ketoneis prepared from methyl-Z-pyridyl ketone with excess of methyliodide inacetone at room temperature, said quaternary pyridine ketone reactedwith hydroxylamine hydrochloride neutralized with sodium hydroxide to pH6-7 to form methyl-Z-pyridyl ketoxime methiodide.

10. A process for producing a new class of oximes of the general formulawherein R is a member selected from the group consisting of H and loweralkyl and R" is a radical selected from the group consisting of methyland ethyl groups and X represents the anionic part of the R"X salt,which comprises reacting a quaternary pyridine aldehyde withhydroxylamine.

No references cited.

1. AS A NEW COMPOSITION OF MATTER, A NEW CLASS OF OXIMES OF THE GENERALFORMULA